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. 2011 May 11;286(28):25065–25075. doi: 10.1074/jbc.M110.214486

FIGURE 4.

FIGURE 4.

Striatin and CCM3 bridge the kinase and phosphatase components of STRIPAK. A, direct in vitro interaction between GST-Mst4 and CCM3. GST-Mst4 kinase or GST alone were incubated with soluble CCM3, and a GST pulldown followed by SDS-PAGE and Coomassie staining was performed. B, CCM3 bridges Mst4 to STRN3 in vitro. GST-Mst4 was incubated with soluble STRN3(1–338) in the absence or presence of soluble CCM3. GST pulldown was followed by SDS-PAGE and Coomassie staining. STRN3(1–338) could be precipitated with GST-Mst4 only when incubated with free CCM3. The soluble lane shows amount of CCM3 and/or STRN3(1–338) in the binding assay. C, depletion of CCM3 or striatins by esiRNA prevents association of the PP2A phosphatase and MST4 kinase. HeLa cells were transfected with the indicated esiRNAs. (Note that the esiRNA mixture for striatins targets all three paralogs.) Luc indicates that a non-targeting esiRNA directed against luciferase was employed; none indicates a mock transfection. After cell lysis (total cell lysate input shown on the left), immunoprecipitation of MST4 (center) or STRN3 (right) was performed using antibodies against the endogenous proteins. SDS-PAGE was followed by immunoblotting using antibodies against the indicated endogenous proteins. Arrows indicate the position of each protein; stars indicate the decrease in intensities of the MST4 and CCM3 bands in the immunoprecipitates, as these proteins migrate close to cross-reacting species in the immunoprecipitates. Depletion of striatins, CCM3, or MST4 all prevent recruitment of PP2Acat and PP2AA to MST4, indicating that CCM3 and striatins are responsible for the interaction between the kinase and the phosphatase components of STRIPAK. STRN association with MST4 is prevented by the depletion of MST4 and CCM3, consistent with the bridge model described in B. Depletion of striatins has no effect on the recruitment of CCM3 to MST4. Although depletion of the striatins alters the recovery of PP2Acat and PP2AA to STRN3, as expected, depletion of CCM3 or MST4 has no effect on these interactions. D, model for the structural organization of core STRIPAK.